The present invention relates to polypeptide antagonists of the human cytokine interleukin 8 or other alpha therapeutic chemokines and to the method of using these antagonists.
Interleukin 8 (IL-8) is a cytokine that promotes the recruitment and activation of neutrophil leulcocytes and represents one of several endogenous mediators of the acute inflammatory response. In the past it was variously termed neutrophil-activating factor, monocyte-derived neutrophil chemotactic factor, interleukin-8 (IL-8), and neutrophil-activating peptide-1. IL-8 has gained the widest acceptance and will be used herein.
The most abundant naturally occurring form of the IL-8 monomer is a 72-residue protein apparently derived by processing of a 99-residue precursor. Other proteins with related sequences, including neutrophil-activating peptide-2 1 ENS-78 and GRO.alpha. (with melanoma growth stimulatory activity) are IL-8 homologues which have neutrophil-activating properties.
IL-8 is a member of the chemokine superfamily that is divided into two distinct function classes: alpha (.alpha.) and beta (.beta.). The members of each class share an organizing primary sequence motif. The .alpha. members are distinguished by a C-X-C motif with the first two cysteines in the motif separated by an intervening residue. C-X-C chemokines are potent chemoattractants and activators for neutrophils, and are represented by IL-8. The .beta. family chemokines have a C--C motif and are equally potent chemoattractants and activators of monocytes. It appears that the two sides of the chemokine family have clearly defined functions: the C-X-C subfamilies cannot activate monocytes while the C--C subfamily has no effect on neutrophils. Nonetheless these two families of chemokines have similar structures although fairly low sequence homology (30 to 35%). Proteins within the same family such as platelet factor four (PF-4) are structurally related to IL-8 (35% sequence identity) but in this example lack the N terminal ELR sequence (Glu-Leu-Arg) which has been shown by site directed mutagenesis to be critical for IL-8 activity and thus, PF-4 has an entirely different profile of activity. Indeed, when the ELR sequence is added to the N-terminus of PF-4 it has been found that the modified protein has potent neutrophil activation and chemoattractant properties (Clarlc-Lewis, I; Dewald, B.; Geiser, T.; Moser, B.; Baggiolini, M.: Platelet factor 4 binds to interleukin 8 receptors and activates neutrophils when its N terminus is modified with Glu-Leu-Arg. Biochemistry 90:3574-3577, 1993). However this may not be true for all of the chemokines since two of the proteins related to IL-8, .gamma. interferon inducible protein (IP-10) and monocyte chemroattractant protein 1 (MCP-1) do not acquire neutrophil activating properties when the ELR structural determinants are added. Interestingly, when the E and the L of the ELk motif are removed from IL-8 the molecule acts as an antagonist for IL-8 (Moser, B.; Dewald, B.; Barella, L.; Schumacher, C.; Baggiolini, M.; Clark-Lewis, Lewis, I: Interleulin-8 antagonists generated by N-terminal modification. J Bio Chem 268:7125-7128, 1993).
In other studies by Clark-Lewis (Clark,Lewis, I., et al.: Structural requirements for interleukin-8 function identified by design of analogs and CXC chemokine hybrids. J Biol Chem 269:16075-16081, 1994) it was shown that conservative substitutions are accepted into the 10-22 region of IL-8 in contrast with the ELR motif (residues 4-6). They concluded that the disulfide bridges and the 30-35 turn provide a structural scaffold for the NH.sub.2 terminal region which includes a primary receptor binding site (ELR motif) and secondary binding and conformational determinants as seen in residues 10 through 22. Other studies using mutants of IL-8 and melanoma growth stimulating activity (MGSA) and recombinant EL-8 .alpha./.beta. receptors stably expressed in human cells demonstrated that there was a second site on the molecule responsible for binding. It appears that the carboxy terminus distal to amino acid 50 is not important in high affinity binding to the .alpha. receptor although both the amino and carboxy termini appear to be important for binding to the .beta. receptor (Schraufstatter, I. S., et al., Multiple sites on IL-8 responsible for binding to .alpha. and .beta. IL-8 receptors. J Immunol 151:6418-6428, 1993). In summary, it appears that there are at least two and maybe three regions responsible for binding on IL-8. Further, the specific contact pharmacophore may vary depending upon whether or not the .alpha. or the .beta. receptor is being examined.
Inflammation and autoimmune responses are initiated by leukocytes which migrate out of the microvasculature and into the extravascular space in response to chemoattractant molecules. These chemoattractants may be from the host and include cytolines, activated complement components or may be released from an invading organism (e.g., N-formylated peptides or MDP dipeptide). Once exposed to chemoattractants within the vasculature, the leukocytes become activated and capable of adhering to the endothelium providing the first step in the development of inflammation. Stimulated neutrophils adhere to the endothelium of the microvasculature in response to a gradient of chemoattractants which direct the cells into the extravascular space toward the source of the chemoattractant. (Anderson et al., J Clin Invest 74:536-551, 1984; Ley, K, et al., Blood 77:2553-2555, 1991; Paulson, J. C., Selectincarbohydrate-mediated adhesion of leukocytes, Adhesion: Its Role in Inflammatory Disease, W. H. Freeman, 1992; Lasky, L. A., The homing receptor (LECAM 1/L-selectin), Adhesion: Its role in inflammatory disease, W. H. Freeman, 1992.)
Bevilacqua et al., (J Clin Invest 76:2003-2011, 1985), demonstrated that cytokines and endotoxin stimulated the endothelium to become more adhesive for leukocytes. Subsequent observations (Buyon, J. P., et al., Clin Immunol Immunopathol 46:141-149, 1988; Abramson, S. B., et al., Hosp Pract 23:45-56, 1988; Clark-Lewis, I. et al., Biochemistry 90:3574-3577, 1993); have suggested that the endothelium has a critical role in the events leading to the development of the inflammatory lesion. This model of inflammation suggests that leukocytes are directed to an inflamed locus by stimulated endothelium. After stimulation with cytolkines or bacterial products, the endothelium arrests leukocytes as they traverse (roll along) the microvasculature near sites of inflammation. After being forced to stop in the microvasculature, the leukocytes are then activated to adhere more tightly to the endothelium and to migrate to the abluminal aspect of the vessel. The leukocyte, once it is out of the blood vessel is then capable of following a gradient of chemoattractants toward the exciting pathogen.
Vascular endothelium, activated by stimulants such as IL-1, IL-8, TNF, or LPS, appears to play a pivotal role in this process through the production of pro-inflammatory substances, including chemoattractants and cytokines such as chemokines.
The inflammatory properties of IL-8 were initially demonstrated from a purified natural product injected intradermally into rabbits to evaluate the proinflammatory properties, (Rampart, M. et al., Am J Path 135(1L):21-25, 1989). More recently neutralizing antibodies to human IL-8 were shown to have a protective effect in inflammatory lung injury in rats. This antibody blocked the glycogen-induced accumulation of neutrophils in rats and was protective against lung interdermal vascular injury induced by the disposition of IgG immune complexes. This latter model of injury has been shown to be E-selectin dependent. The protective effect of the neutralizing antibody correlated with reduced tissue accumulation of neutrophils as measured by myeloperoxidase content. Preliminary nonhuman primate studies have confirmed the activity of IL-8 on hematological parameters. IL-8 was administered by both bolus and continuous infusion to baboons. This resulted in a rapid, transient and severe granulocytopenia followed by granulocytosis that persisted as IL-8 levels remained detectable within the circulation. Histopathological examination revealed a mild to moderate neutrophil margination in the lung, liver and spleen which was of greater severity in animals receiving the continuous infusion of IL-8.
High levels of intravascular IL-8 have been reported in systemic conditions such as septic shock (Danner, R. L., et al., Clin Res 38:352A, 1990). These authors have speculated that intervascular IL-8 may impair leukocyte adhesion and thus protect organs from PMN mediated injury. The intravenous administration of IL-8 induced an immediate and transient neutropenia that was similar in kinetic profile to that described with other chemoattractants. This neutropenia was a result of pulmonary PMN sequestration and is consistent with the demonstration of abundant IL-8 receptor on PMNs. Following this transient neutropenia (approximately 30 minutes) cells recirculate with a normal half life. Shortly thereafter neutrophilia, a characterisic of IL-8, is observed. The neutrophilia likely reflects recruitment of mature PMNs to a marginal pool in the lung and other organs as well as immature PMNs from the marrow.
Endothelial cells can exert both proinflammatory and anti-inflammatory effects by virtue of the mediators they generate. Endothelial cells can be stimulated to generate IL-8, but unlike other mediators, IL-8 may be released from the endothelial cell. Alternatively, endothelial cell produced IL-8 is an important chemoattractant and activator of neutrophils. There is evidence that systemic IL-8 can bind to endothelial cells which could produce a local activation of the endothelium resulting in the ability of this altered endothelium to attract neutrophils that have come into contact with the (activated) endothelium. One working hypothesis is that IL-8 initially functions as a proinflammatory cytoline, whereas its continued generation and release from the endothelium ultimately causes a down regulation of neutrophils, with a curtailment in their further recruitment. Whether the cell associated IL-8 or released IL-8 provides the vital contribution to the outcome of the inflammatory response remains unresolved.
IL-8 receptors are "promiscuous" and respond with a calcium flux when bound by structurally related ligands with the following order of potency: IL-8&gt;MGSA&gt;NAP-2 which correlates with the effectiveness of these compounds when competing with the radio labeled IL-8 for binding to neutrophils C5a, a structurally related chemoattractant that is similar in size and charge to IL-8 and which has a receptor in the same family, does not activate the IL-8 receptor.
The in vitro effects of IL-8 on neutrophils are similar to those of other chemotactic antagonists such as C5a and fMet-Leu-Phe and include induction of a transient rise in cytosolic free calcium, the release of granules containing degradative enzymes such as elastase, the respiratory H.sub.2 O.sub.2 burst, neutrophil shape change, and chemotaxis. IL-8 appears to bind to two or more receptor sites on neutrophils with a frequency of approximately 64,000/cell and a K.sub.d of 0.2 nM.
The three-dimensional structure of IL-8 is known by two-dimensional NMR and x-ray diffraction techniques. The IL-8 monomer has antiparallel .beta. strands followed by a single overlying COOH-terminal .alpha. helix. Two disulfide bridges, between cysteines 7 and 34, and between cysteines 9 and 50 seem to stabilize the tertiary structure. Residues 1-6 and the loop residues 7-18 seem to have little defined secondary structure. En solution, IL-8 is a noncovalent homodimer which is stabilized primarily by interactions between the .beta. strands of the two monomers.
Examination of the three-dimensional structure indicates that following the cysteine at position 50, the residues form a type 1.beta. turn (at residues 51 to 55) followed by an amphipathic .alpha. helix (at residues 55 to 72) that transverses the .beta. sheet. The hydrophobic face of the .alpha. helix interacts with and stabilizes the hydrophobic face of the .beta. sheet. Some of the interactions are between the two subunits of the dimeric molecule.
As it is established that IL-8 is a key mediator of inflammatory diseases, it would be desirable to identify substances capable of blocking or interrupting the activity of IL-8 for use in anti-inflammatory compositions. Such compositions may prove to be advantageous over presently available NSAID's, steroid based anti-inflammatory drugs and cytotoxic drugs which often have severe side-effects with the continued usage that is required for chronic inflammatory diseases. It would also be desirable to identify IL-8 analogs having an increased inflammatory activity for medical research applications.
IL-8 has been previously produced through chemical synthesis (for example see: Clark-Lewis, et al., "Chemical Synthesis, Purification, and Characterization of Two Inflammatory Proteins; Neutrophil-Activating Peptide-1 (Interleuldn-8) and Neutrophil-Activating Peptide-2" (1991) Biochemistry 30: 3128-3135) and by recombinant DNA methods (for example see: Herbert, et al., "Scanning Mutagenesis of Interleukin-8 Identifies A Cluster of Residues Required for Receptor Binding" (1991) J. Biol. Chem. 286: 18989-18994). In addition, it is known that IL-8 exists in several forms that vary at the NH.sub.2 -terminus, which have been detected in preparations purified from natural sources. These variations correspond to the predominant 72-residue form (which is generally considered to be the prototype IL-8 molecule); a 77-residue form having 5 additional NH.sub.2 -terminus amino acids on each monomer; and, two shortened forms having residues 3-72 and 4-72 of the 72 amino acid form, respectively.